Monitoring extracellular glutamate in the rat brain by microdialysis and microsensors van de Zeyden, Miranda

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1 University of Groningen Monitoring extracellular glutamate in the rat brain by microdialysis and microsensors van de Zeyden, Miranda IMPORTANT NOTE: You are advised to consult the publisher's version (publisher's PDF) if you wish to cite from it. Please check the document version below. Document Version Publisher's PDF, also known as Version of record Publication date: 211 Link to publication in University of Groningen/UMCG research database Citation for published version (APA): van de Zeyden, M. (211). Monitoring extracellular glutamate in the rat brain by microdialysis and microsensors: pharmacological applications Groningen: s.n. Copyright Other than for strictly personal use, it is not permitted to download or to forward/distribute the text or part of it without the consent of the author(s) and/or copyright holder(s), unless the work is under an open content license (like Creative Commons). Take-down policy If you believe that this document breaches copyright please contact us providing details, and we will remove access to the work immediately and investigate your claim. Downloaded from the University of Groningen/UMCG research database (Pure): For technical reasons the number of authors shown on this cover page is limited to 1 maximum. Download date:

2 Chapter 5 Are brain extracellular glutamate levels recorded by microdialysis related to synaptic events? Abstract INTRODUCTION MATERIALS AND METHODS Animals Materials (drug treatment and doses) Surgery and brain dialysis probes Microdialysis Experiments Chemical assays Expression of results and statistics RESULTS Basal values Effect of infusion of different drugs into the ventral tegmentum area (VTA) on the dialysate content of dopamine in the ipsilateral nucleus accumbens (NAc) DISCUSSION Infusion of glutamate or blocking high affinity glutamate transporters (by infusing TBOA or TBOA combined with glutamate) Modifying astrocytic glutamate release by blocking glutamine synthesis Modifying the glutamate-cystine exchanger Modifying mglur2/3 receptors by infusing mglur specific compounds CONCLUSION

3 Chapter 5 Abstract In the present study a dual-probe microdialysis approach in the mesolimbic dopamine pathway was used to distinguish glutamate s role as a neurotransmitter from its role in non-neuronal events. In this model one probe is inserted into the dopamine cell body region (the VTA) and a second probe is inserted into the axon terminal region of the ipsilateral NAc. Glutamate levels in the VTA are modified by infusing glutamate related compounds in the first probe, whereas their effects on dopamine neurons are recorded by dialysate levels of dopamine in from the second probe. By monitoring glutamate in the VTA and dopamine in the ipsilateral NAc, the correlation between extracellular glutamate levels in the VTA (as measured in dialysates) and functional effects exerted on VTA dopamine neurons can be studied. Infusion of glutamate showed a clear mismatch between extracellular levels of glutamate in the VTA and the absence of effects on the dopamine cells in the VTA (as measured by dopamine in the NAc). However it was demonstrated that glutamate levels enhanced by the transporter blocker TBOA were able to reach synaptic receptors. Even so exogenously administered glutamate in the presence of TBOA - could at least partly reach the synaptic glutamate receptors and modify dopamine neurons. When extracellular glutamate was strongly reduced by MSO infusion, no effect was seen on dopamine release in the NAc. From this observation we may hypothesize that an important part of the glutamate sampled by microdialysis is derived from glial cells and do not relate directly to synaptic events. Experiments aimed to identify role of the cystineglutamate exchanger to modifying glutamate levels, indicated that a minor part of extracellular glutamate in the NAc might indeed be regulated by this antiporter. Interestingly this glutamate pool was able to affect the activity of the dopamine neurons. Experiments using mglu2/3r specific compounds indicated that a small but significant pool of glutamate - detectable in dialysates - was able to affect the activity of the dopamine neurons. Taken together, these observations suggest that care should be taken to relate glutamate in microdialysates to neuronal glutamatergic activity in the brain. However during certain pharmacological conditions a minor part of glutamate sampled by microdialysis might be directly related to synaptic events.

4 Are brain extracellular glutamate levels recorded by microdialysis related to synaptic events? 1. INTRODUCTION Glutamate, the major excitatory neurotransmitter, mediates 9% of the excitatory neurotransmission in the central nervous system (Cotman and Monaghan 1986). Central glutamate pathways are associated in a wide range of normal brain functions including cognition, memory and learning, plasticity and motor movement. Alternations in glutamate transmission are implicated in pathologies ranging from neurotoxicity to neuropsychiatric disorders (Gardoni and Di Luca 26), including stress, substance abuse and addiction, schizophrenia, stroke and epilepsy. Explicit information about glutamate in the extracellular space of living brain tissue would contribute significantly to the fundamental understanding of the role of glutamate in these disorders. Currently the most common technique employed to study glutamate in the intact mammalian brain is in vivo microdialysis. However the synaptic contribution to extracellular glutamate is difficult to estimate and matter of debate, as basal and many pharmacological induced glutamate levels in microdialysates are (in contrast to most neurotransmitters) not tetrodotoxin (TTX)- or calcium dependent (Timmerman and Westerink, 1997). In the present study we have used the mesolimbic dopamine system as an indirect in vivo model to interpret extracellular glutamate and the influence of pharmacological compounds on its synaptic activity. Glutamate afferents in the ventral tegmental area (VTA) are found to play an important role in regulating the activity of dopamine neurons (Takahata and Moghaddam 2). To evaluate the effect of glutamate on dopamine neurons we have used a dual-probe microdialysis method with one probe located in the VTA and the second probe in the ipsilateral nucleus accumbens (NAc) (Westerink et al. 1996). We manipulated the glutamatergic afferent input to the dopamine neurons in the VTA by infusing pharmacological active compounds via a microdialysis probe (probe 1), while concurrently recording the effect of this modulation on the output of the dopamine neurons as determined by the release of dopamine in the ipsilateral NAc (probe 2). In addition we have also used probe 1 to measure the modified extracellular glutamate levels in the VTA. By comparing glutamate changes in probe 1 and dopamine changes in probe 2 we are able to relate changes in microdialysate glutamate concentrations to synaptic glutamatergic effects. 11

5 Chapter 5 The influences of the following pharmacological compounds, supposed to affect glutamate levels in the synaptic cleft, were determined during infusions in probe 1: i. infusing glutamate ii. blocking high affinity glutamate transporters by infusing TBOA 1 or TBOA with glutamate iii. altering astrocytes function by inhibiting glutamine and subsequently glutamate synthesis by infusing MSO 2 iv. blocking low affinity glutamate transporter mechanisms such as cystineglutamate exchange by infusing HCA 3 or CPG 4 v. modifying mglur2/3 receptors that are known to modulate glutamate release, by infusion of the agonist APDC 5 or antagonist APICA 6 1 DL-threo-ß-Benzyloxyaspartic acid (TBOA) 2 L-Methionine sulfoximine (MSO) 3 L-Homocysteic acid (HCA) 4 (S)-4-Carboxyphenylglycine (CPG) 5 (2R,4R)-4-Aminopyrrolidine-2,4,dicarboxylate (APDC) 6 (RS)-1-Amino-5-phosphonoindan-1-carboxylic acid (APICA) 12

6 Are brain extracellular glutamate levels recorded by microdialysis related to synaptic events? 2. MATERIALS AND METHODS 2.1 Animals Male albino rats of a Wistar-derived strain ( g; Harlan, Zeist, The Netherlands) were used for the experiments. The rats were housed individually in plastic cages (35x35x4 cm) with food and water ad libitum. Animals were kept on a 12 h light schedule (from 7 A.M. until 7 P.M). The experiments were concordant with the declaration of Helsinki and were approved by the Animal Care Committee of the Faculty of Mathematics and Natural Sciences of the University of Groningen, The Netherlands. 2.2 Materials (drug treatment and doses) The following drugs were used: DL-threo-b-Benzyloxyaspartic acid (DL-TBOA), (S)-4- Carboxyphenylglycine (CPG), (RS)-1-Amino-5-phosphonoindan-1-carboxylic acid (APICA), (2R,4R)-4-Aminopyrrolidine-2,4,dicarboxylate (APDC) (purchased from Tocris Neuramin, Essex, England). Glutamate, L-Homocysteic acid (HCA), L-Methionine sulfoximine (MSO), Tetrodotoxin (TTX) (purchased from Sigma-Aldrich B.V., Zwijndrecht, the Netherlands). The drugs were first dissolved in water at a concentration of 1 µm and further diluted with Ringer. The composition of the Ringer s solution was (in mm) NaCl 147., KCl 3., CaCl and MgCl Drugs dissolved in Ringer were infused via retrograde microdialysis into the VTA. One of the conditions of the dual-probe technique is that infused concentrations of drugs should be sufficiently high to block or stimulate the appropriate receptors in the infused area (here aimed at the VTA). Infused doses were based on earlier studies on related experiments and according to their IC 5 or EC 5 values as described previously by various investigators (Shimamoto et al. 1998;Shoepp et al. 1999). 2.3 Surgery and brain dialysis probes Microdialysis was performed with two I-shaped microdialysis probes constructed with a Regenerated cellulose or Hospal AN67HF: polyacrylnitrile dialysis fibre membrane (Brainlink, Groningen, The Netherlands), with an i.d 22 µm and o.d 31 µm. One probe (exposed length 1.5 mm) was implanted into the VTA, and the second probe (exposed length 1.5 mm) was implanted into the ipsilateral NAc. Drugs were infused into the brain via the VTA probe, and the NAc probe was used to record extracellular dopamine. Coordinates of the implantation were as follows: A/P 2.2, L/M -1.6 and V/D -7.2 (NAc); A/P -5., L/M +.9, and V/D -8. (VTA), from bregma point and dura. The dialysis probes were stereotactically implanted in the animal brain under the following conditions: isoflurorane 2%, O 2, 6 ml/min and local bupivacaine hydrochloride monohydraat (marcaine,.5% adrenaline) anaesthesia. The microdialysis probes were permanently 13

7 Chapter 5 fixed to the skull using stainless steel screws and methylacrylic cement. Rats were placed in individual Perspex cages (35x35x4 cm) where they had free access to food and water. Animals were allowed to recover 24 hr before microdialysis experiments commenced. 2.4 Microdialysis Experiments Microdialysis experiments were carried out 24 to 72 hr after implantation of the probes. In all experiments the dialysis probes were perfused with a Ringer solution at a flow rate of 1.5 µl/min with aid of a pump Technical and Scientific Equipment, Bad Homburg, Germany. All pharmacological agents used for infusion were prepared in this Ringer solution as described. All inlet and outlet tubing was from flexible PEEK (I.D..15 mm; Watson-Marlow). An on-line approach was used to determine dopamine in the NAc where the dialysate was on-line collected into an HPLC injection loop (3 µl) and automatically injected every 15 min. An off-line approach was used to determine glutamate in the VTA. Microdialysis samples were collected every 15 min in HPLC vials containing 7.5 µl.2 M acetic acid to prevent amino acid degradation. The collected samples were stored in a freezer at - 8ºC. After 4 basal samples were collected, drug infusion was performed for the duration of the experiment, followed by reperfusion of the Ringer solution. Implantation of the cannulas was evaluated functionally as described below. The experiments were finished with infusion of 5 µm baclofen or 1 µm TTX into the VTA probe, and the response in the NAc was determined. A decrease in extracellular dopamine in the NAc to at least 4% of controls was considered as a measure for correct implantation. When the experiment was terminated, the animals were anaesthetized with isofluraan and than euthanized with 1. mg/kg sodiumpentobarbital. The brain was fixed with 4% paraformaldehyde. Coronal sections (4 µm thick) were made, and dialysis probes placement verified according to the atlas of (Paxinos and Watson 1986). 2.5 Chemical assays Dopamine (DA), dopamine, 3,4-dihydroxyphenylacetic acid (DOPAC) and 5- hydroxyindoleacetic acid (HIAA) was quantified by HPLC coupled with electrochemical detection. A Shimadzu (LC-1AD) pump was used in conjunction with an electrochemical detector (ESA Coulochem II), with a Coulochem 511 detector cell, potential first cell: +5 mv; potential second cell: -25 mv). A reverse-phase column (15 x 4.7 mm 2 ; Supelco LC18, Bellefonte, PA) was used. The mobile phase consisted out of mixture of 14

8 Are brain extracellular glutamate levels recorded by microdialysis related to synaptic events?.5 M sodium acetate, 1 M octanesulfonic acid,.5 mm Na 2 -EDTA, and 13 ml/l methanol adjusted to ph 4.2 with acetic acid, at a flow of 1. ml/min. The detection limit of the assay was ±3 fmol/sample (on-column). Glutamate in the microdialysis samples were detected with high performance liquid chromatography with tandem mass spectrometry (LC-MS/MS). In brief, 1 µl samples were mixed with internal standard,1.2 µm [D5]-glutamate (from Cambridge Isotope Laboratories Inc., Andover, USA) and 3 nm [D6]-GABA (from Isotec, Miamisburg, USA), and were automatically derivatized with SymDAQ (Symmetrical DiAldehydes Quaternary ions) in the autosampler (SIL-1AD vp, Shimadzu, Japan) by adding 4 µl reagent to the sample. After a reaction time of 2 minutes, 4 µl of the mixture was injected into the LC system by an automated sample injector (SIL-1AD vp, Shimadzu, Japan). Chromatographic separation was performed on a reversed phase Hypersil Gold 5 x 2.1 mm (1.9 µm particle size) held at a temperature of 4 C. Components were separated using a linear gradient of ACN /.1% FA in ultrapure water /.1% FA (flow rate.3 ml/min) according to the following scheme: LC time scheme. Time (min) % ACN/.1 % FA. 3; ; 4.5 9; After 2 min of eliminating the waste, the flow of LC was switched to the MS for the detection of glutamate. MS analyses were performed with an API 4 MS/MS system consisting of an API 3 MS/MS detector and a Turbo Ion Spray interface (both from Applied Biosystems, the Netherlands). The acquisitions were performed in positive ionization mode, with ion spray voltage set at 4 kv with a probe temperature of 2 C. The instrument was operated in multiple-reaction-monitoring (MRM). Data were calibrated and quantified using the Analyst tm data system (Applied Biosystem, version 1.4.2). 2.6 Expression of results and statistics All values given are expressed as percent of the baseline. The average concentration of four consecutive base-line samples (less than 1% variation) was considered the basal level and was set as %. Statistical analysis (SPSS14. for windows) was performed using one-way analysis of variance for repeated measures followed by the post-hoc Dunnett s multiple comparison testing. The level of significance was set at p<.5. 15

9 Chapter 5 3. RESULTS 3.1 Basal values Mean basal values (± SEM 7 ) were for DA 3.56 ±.43 fmol/min and for its metabolite DOPAC and the serotonin metabolite 5-HIAA (± S.E.M).72 ±.6 and.24 ±.2 pmol/min respectively (n=5). 5-HIAA levels are usually not related to dopamine metabolism and can be considered as an internal standard representing the stability of the microdialysis experiments. Table 1: Summary of the pharmacological agents infused into the ventral tegmental area (VTA) and basal extracellular dopamine levels measured in the nucleus accumbens (NAc) in vivo with dual probe microdialysis. Values are given as data SEM. Drug Mechanism of action Dose Basal DA Basal DOPAC Basal HIAA n um fmol/sample pmol/sample pmol/sample TTX Na + channel antagonist ± ± ±.7 6 GLU Excitatory neurotransmitter ± ± ± TBOA EAAT1 antagonist ± ±.13.2 ±.2 6 EAAT2 antagonist EAAT3 antagonist TBOA & Excitatory neurotransmitter 2.84 ± ± ±.6 6 GLU and EAAT1-3 antagonist & & 1 MSO Glutamine synthetase ± ± ±.9 4 inhibitor 5 25 HCA NMDA agonist X - C antagonist ± ±.4.28 ±.5 6 CPG mglur-1/5 antagonist ± ± ±.6 6 X - C antagonist 5 5 APICA mglur-2/3 antagonist ± ± ±.8 5 APDC mglur-2/3 agonist ±.3.48 ± ± Standard error of the mean (SEM) 16

10 Are brain extracellular glutamate levels recorded by microdialysis related to synaptic events? 3.2 Effect of infusion of different drugs into the ventral tegmentum area (VTA) on the dialysate content of dopamine in the ipsilateral nucleus accumbens (NAc) Effect of infusion of TTX into the VTA on the dialysate content of dopamine in the ipsilateral NAc Perfusion of TTX (1 µm) through the VTA microdialysis probes decreased extracellular dopamine in the ipsilateral NAc in all animals. Dopamine levels were reduced to nondetectable levels and remained low throughout the experiment (Figure 1). This decrease was statistically significant (F 1.53 = ; p<.) (n=6). This effect can be used as a functioned test of proper implantation of the probe. The TTX effect demonstrates that the two probes are properly implanted with respect to the mesolimbic dopaminergic pathway. This decrease was previously established in dual-probe microdialysis studies (Schilström et al. 1998;Mathé et al. 1999;Legault et al. 2). Rats showed turning behaviour 3 minutes after TTX infusion. Effect of infusion of glutamate into the VTA on the dialysate content of glutamate in the VTA and dopamine in the ipsilateral NAc Exogenous infusion of glutamate at a concentration of 1 µm to 1 mm in the VTA increased glutamate concentrations significantly (F 4,111 =31.2; p<., n=5) (Figure 2). However exogenous infusion of glutamate at concentrations of 1 µm to 1 mm in the VTA did not affect the levels of dopamine measured in the ipsilateral NAc (F 4,139 =1.884; p=.117, n=5) (Figure 3). Effect of infusion of TBOA alone and combined with Glutamate into the VTA on the dialysate content glutamate in the VTA and dopamine in the ipsilateral NAc The broad-spectrum inhibitor of Na + -dependent glutamate transport TBOA (Shimamoto et al. 1998) was infused into the VTA at increasing concentrations of 1,, 3, 5 µm and 1 mm for two hours at each concentration. These dosages were not all administer to the same animals, due to practical reasons. The combined effect of TBOA on glutamate concentrations in the VTA is shown in Figure 4a and on dopamine concentrations in the NAc in Figure 4b. In response to this, a concentration dependent increase of ±15%, ±281% and ±518% in extracellular glutamate was seen in the VTA when 1, µm and 1 mm were infused, which was found to be significant for µm and 1 mm (F 3,167 =46.12; p<., n=4). Using, 3 and 5 µm TBOA a concentration dependent increase of ±255%, 17

11 Chapter 5 ±399% and ±436% respectively was observed, and was found to be significant at all concentrations (F 3,87 =7.941; p<., n=4) (Figure 4a). The extracellular dopamine in the ipsilateral NAc increased to ±96%, ±17% and ±153% of baseline when 1, µm and 1 mm TBOA was infused into the VTA, which was found to be significant for 1mM (F 3,167 =46.12; p<., n=6) (Figure 5a). Using, 3 and 5 µm TBOA a concentration dependent increase of ±14%, ±129% and ±155% respectively was observed, and was found to be significant at a concentration of 3 and 5 µm(figure 5b). The combined infusion of µm TBOA with 1mM and 1mM exogenous glutamate respectively caused a significant increase in glutamate concentrations in the VTA (F 3,87 =39.153; p<., (n=4) (Figure 6). The combined infusion of µm TBOA with 1mM and 1mM exogenous glutamate respectively caused a significant increase in the extracellular dopamine concentrations in the ipsilateral NAc (F 3,131 = ; p<., n=6). After 9 minutes infusion of µm TBOA the co-administration of 1mM and 1mM glutamate increased extracellular dopamine levels significantly to ±125% and ±136% respectively (Figure 7). Administration of DL-TBOA into the VTA induced behavioural changes commonly associated with the administration of glutamate receptor agonists, such as NMDA and quinolinic acid. This issue is addressed in discussion. Effect of infusion of MSO into the VTA on the dialysate content of glutamate in the VTA and dopamine in the ipsilateral NAc Infusion of glutamate synthetase inhibitor methionine sulfoximine (MSO) into the VTA (1 mm and 5 mm, 2 hours each) decreased extracellular glutamate levels in the VTA significantly (F 2,39 =6.62; p=.5, n=4) (Figure 8). MSO decrease dopamine concentrations in the ipsilateral NAc not significantly (F 2,39 =15.239; p=.924, n=4) (Figure 9). Effect of infusion of cystine, HCA or CPG into the VTA on the dialysate content of glutamate in the VTA and dopamine in the ipsilateral NAc During infusion of 1 µm and 1 mm cystine into the VTA, extracellular glutamate concentrations increased in the VTA to ±18% and ±196% respectively and extracellular dopamine concentrations increased in the NAc to ±126% and ±131% respectively. These increases were found to significant in both the VTA (F 2,63 =3.734; p<., n=5) and NAc (F 2,95 =13.476; p<., n=6) (Figure 1 and 11) 18

12 Are brain extracellular glutamate levels recorded by microdialysis related to synaptic events? The infusion of the cystine-glutamate exchange inhibitor HCA in the VTA caused a significant decrease in extracellular glutamate concentrations in the VTA, as a concentration of 5 µm caused a decrease to ±74% of basal levels (F 3,11 =5.86; p=.2, n=5) (Figure 12). HCA had no significant effect on dopamine release in the NAc (F 3,131 =.19; p=.955, n=6) (Figure 13). The reverse dialysis of the cystine-glutamate exchange inhibitor CPG in the VTA caused a significant decrease to ±85% of control after infusion of 5 µm, however a concentration of 5 and 5 µm increased glutamate levels (±134%), which was however not significant compared to basal levels in the post hoc test (F 3,19 =4.348; p=.6, n=5) (Figure 14). CPG infused in the VTA at a concentration of 5 µm, 5 µm and 5 mm caused an increase in extracellular dopamine of ±119%, ±134% and ±147% respectively which was found to be significant for each concentration (F 3,131 =11.5; p<., n=6) (Figure 15). Effect of infusion of APDC or APICA into the VTA on the dialysate content of glutamate in the VTA and dopamine in the ipsilateral NAc Infusion of the mglur II agonist APDC in the VTA, at a concentration of 5, 5, 5 µm and 5 mm for 9 minutes induced a decrease in extracellular glutamate levels to 16%, 93%, 79% and ±72% respectively in the VTA which was significant at the highest concentration (F 4,167 =4.42; p=.4, n=6) (Figure 16). In the NAc APDC induced a slight but significant increase in extracellular dopamine to ±16% after exposure to 5 µm APDC, and showed a significant decrease to ±83% of dopamine basal levels in the ipsilateral NAc after exposure to 5 mm APDC (F 4,139 =6.66; p<., n=5) (Figure 17). Infusion of the mglur II antagonist APICA, at a concentration of 1, µm and 1 mm for 9 minutes each in the VTA showed a significant increase in extracellular glutamate levels (±164%) in the VTA, (F 3,196 =5.324; p=.49, n=7) (Figure 18). APICA induced a significant increase of extracellular dopamine values in the ipsilateral NAc. Infusion of µm and 1 mm APICA in the VTA induced an increase to ±118% and ±146% respectively. Statistics for concentrations are significant (F 3,139 =55.13; p<., n=5) (Figure 19). 19

13 Chapter DA DOPAC HIAA 12 % of Baseline uM TTX infusion Time (min) Figure 1: Effect of infusion of TTX (1 µm) into the VTA on the extracellular concentrations of dopamine the ipsilateral NAc. The horizontal bar represents the period of infusion of TTX. Data are given as percentage of basal values ± SEM (n=6). indicates post-hoc significance (p<.5) versus baseline. 11

14 Are brain extracellular glutamate levels recorded by microdialysis related to synaptic events? 6 5 Glutamate release as % of baseline baseline 1uM um 1mM 1mM Exogenous glutamate concentrations infused into the VTA Figure 2: Effect of infusion of glutamate (1µM, µm, 1 mm and 1 mm) into the VTA on the extracellular concentrations of glutamate. Data are given as percentage of basal values ± SEM (n=5). indicates post-hoc significance (p<.5) versus baseline. 12 Dopamine release as % of baseline baseline 1uM um 1mM 1mM Exogenous glutamate concentrations infused into the VTA Figure 3: Effect of infusion of glutamate (1µM, µm, 1 mm and 1 mm) into the VTA on the extracellular concentrations of dopamine the ipsilateral NAc. The horizontal bar represents the period of infusion of glutamate. Data are given as percentage of basal values ± SEM (n=5). 111

15 Chapter Glutamate release as % of baseline control 1uM um 3uM 5uM 1mM TBOA concentrations infused into the VTA Figure 4a: Effect of infusion of TBOA (1,, 3, 5 and µm) into the VTA (bars) on the extracellular concentrations of glutamate. Data are given as percentage of basal values ± SEM (n=4). indicates post-hoc significance (p<.5) versus baseline Dopamine rlease as % of baseline control 1uM um 3uM 5uM 1mM TBOA concentration infused into the VTA Figure 4b: Effect of infusion of TBOA (1,, 3, 5 and µm) into the VTA (bars) on the extracellular concentrations of dopamine in the ipsilateral NAc. Data are given as percentage of basal values ± SEM. indicates post-hoc significance (p<.5) versus baseline. 112

16 Are brain extracellular glutamate levels recorded by microdialysis related to synaptic events? 2 18 DA DOPAC HIAA % of Baseline Dopamine release as % of baseline µm TBOA infusion µm TBOA infusion 1 mm TBOA infusion Time (min) baseline 1uM um 1mM TBOA concentrations infused into the VTA Time: F(27.167)=6.539 p=. Concentration: F(3.167)= p=. Figure 5a: Effect of infusion of TBOA (1, µm and 1mM) into the VTA (bars) on the extracellular concentrations of dopamine the ipsilateral NAc. The horizontal bar represents the period of infusion of TBOA. Data are given as percentage of basal values ± SEM (n=6). indicates post-hoc significance (p<.5) versus baseline. 2 2 DA % of Baseline DOPAC HIAA Dopamine concentration as % of baseline uM TBOA infusion 5uM TBOA infusion 2 um TBOA infusion Time (min) baseline um 3uM 5uM TBOA concentrations infused into the VTA Time: F(21.87)=.982 p=.496 Concentration: F(3,87)=7.941 p=. Figure 5b: Effect of infusion of TBOA (, 3, 5 µm) into the VTA (bars) on the extracellular concentrations of dopamine the ipsilateral NAc. The horizontal bar represents the period of infusion of TBOA. Data are given as percentage of basal values ± SEM (n=4). indicates post-hoc significance (p<.5) versus baseline. 113

17 Chapter Glutamate release as % of control baseline um TBOA um TBOA & 1mM GLU Concentrations infused into the VTA um TBOA & 1mM GLU Figure 6: Effect of infusion of TBOA ( µm) or TBOA with glutamate ( µm TBOA with 1 mm glutamate; µm TBOA with 1 mm glutamate) into the VTA (bars) on the extracellular concentrations glutamate. Data are given as percentage of basal values ± SEM (n=4). indicates post-hoc significance (p<.5) versus baseline. % of Baseline DA DOPAC HIAA # # # # #, Dopamine release as % of baseline um TBOA um TBOA & 1mM GLU um TBOA & 1mM GLU Time (min) baseline um TBOA um TBOA & 1mM Glu Concentrations infused into the VTA um TBOA & 1mM Glu Time: (F 22,11 =19.736; p<.) Concentration: (F 3,131 = ; p<.) Figure 7: Effect of infusion of TBOA ( µm) or TBOA with glutamate ( µm TBOA with 1 mm glutamate; µm TBOA with 1 mm glutamate) into the VTA on the extracellular concentrations of dopamine the ipsilateral NAc. The horizontal bar represents the period of infusion of TBOA. Data are given as percentage of basal values ± SEM (n=6). indicates post-hoc significance (p<.5) versus baseline. 114

18 Are brain extracellular glutamate levels recorded by microdialysis related to synaptic events? 12 Glutamate release as % baseline baseline 1mM 5mM MSO concentrations infused into the VTA Figure 8: Effect of 2 hour infusion of each MSO concentration (1 and 5 mm) in the VTA (bars) on the extracellular concentrations of glutamate. Data are given as percentage of basal values ± SEM (n=4). indicates post-hoc significance (p<.5). 12 Dopamine release as % of baseline baseline 1mM 5mM MSO concentrations infused into the VTA Figure 9: Effect of 2 hour infusion of each MSO concentration (1 and 5 mm) in the VTA (bars) on the extracellular concentrations of dopamine the ipsilateral NAc. The horizontal bar represent the periods of infusion for MSO. Data are given as percentage of basal values ± SEM (n=4). 115

19 Chapter Glutamate release as % of baseline 15 5 baseline um 1mM Cystine concentrations infused into the VTA Figure 1: Effect of cystine ( µm and 1 mm), infused by retrograde dialysis into the VTA, on extracellular levels of glutamate. Data are given as percentage of basal values ± SEM (n=4) indicates post-hoc significance (p <.5) versus baseline. 15 Dopamine levels as % of baseline 5 baseline 1uM 1mM Cystine concentrations infused into the VTA Figure 11: Effect of cystine ( µm and 1 mm) infused by retrograde dialysis into the VTA, on extracellular levels of dopamine in the NAc. The horizontal bar represents the period of infusion of cystine. Data are given as percentage of basal values ± SEM (n=6). indicates post-hoc significance (p<.5) versus baseline. 116

20 Are brain extracellular glutamate levels recorded by microdialysis related to synaptic events? 12 Glutamate release as % baseline baseline 5uM 5uM 5uM HCA concentrations infused into the VTA Figure 12: Effect of 9 minute infusion of each HCA concentration (5, 5 and 5µM) into the VTA (bars) on the extracellular concentrations of glutamate. Data are given as percentage of basal values ± SEM (n=4). indicates post-hoc significance (p<.5) versus baseline. 12 Dopamine release as % of baseline baseline 5uM 5uM 5uM HCA concentrations infused into the VTA Figure 13: Effect of 9 minute infusion of each HCA concentration (5, 5 and 5µM) into the VTA (bars) on the extracellular concentrations of dopamine the ipsilateral NAc. The horizontal bar represents the period of infusion of HCA. Data are given as percentage of basal values ± SEM (n=6). indicates post-hoc significance (p<.5) versus baseline. 117

21 Chapter Glutamate relase as % of baseline baseline 5uM 5uM 5uM CPG concentration infused into the VTA Figure 14: Effect of 9 minute infusion of each CPG concentration (5, 5 and 5µM) into the VTA (bars) on the extracellular concentrations of glutamate. Data are given as percentage of basal values ± SEM (n=4). indicates post-hoc significance (p<.5) versus baseline. Dopamine release as % of baseline baseline 5uM 5uM 5uM CPG concentrations infused in the VTA Figure 15: Effect of 9 minute infusion of each CPG concentration (5, 5 and 5 µm) into the VTA (bars) on the extracellular concentrations of dopamine the ipsilateral NAc. The horizontal bar represents the period of infusion of CPG. Data are given as percentage of basal values ± SEM (n=6). indicates post-hoc significance (p <.5) versus baseline. 118

22 Are brain extracellular glutamate levels recorded by microdialysis related to synaptic events? 12.. Glutamate release as % of baseline baseline 5uM 5uM 5uM 5mM APDC concentrations infused into the VTA Figure 16: Effect of 9 minute infusion of each APDC concentration (5, 5 and 5 µm and 5 mm) in the VTA (bars) on the extracellular concentrations of glutamate. Data are given as percentage of basal values ± SEM (n=6). indicates post-hoc significance (p <.5) versus baseline. 12 Dopamine release as % of baseline baseline 5uM 5uM 5uM 5mM APDC concentrations infused into the VTA Figure 17: Effect of 9 minute infusion of each APDC concentration (5, 5 and 5 µm and 5 mm) in the VTA (bars) on the extracellular concentrations of dopamine the ipsilateral NAc. The horizontal bar represents the period of infusion of APDC. Data are given as percentage of basal values ± SEM (n=5). indicates post-hoc significance (p<.5) versus baseline. 119

23 Chapter 5 2 Glutamate release as % baseline 15 5 baseline 1uM um 1mM APICA concentrations infused into the VTA Figure 18: Effect of 9 minute infusion of each APICA concentration (1 µm, µm and 1 mm) in the VTA (bars) on the extracellular concentrations of glutamate. Data are given as percentage of basal values ± SEM (n=7). indicates post-hoc significance (p<.5) versus baseline Dopamine release as % of baseline baseline 1uM um 1mM APICA concentrations infused into the VTA and NAc Figure 19: Effect of 9 minute infusion of each APICA concentration (1 µm, µm and 1 mm) in the VTA on the extracellular concentrations of dopamine the ipsilateral NAc. Data are given as percentage of basal values ± SEM (n=5). indicates post-hoc significance (p<.5) versus baseline. 12

24 Are brain extracellular glutamate levels recorded by microdialysis related to synaptic events? 4. DISCUSSION Contrary to the growing body of literature regarding in vivo studies of glutamate many questions have remained unanswered regarding the significance of the exact resting concentrations of glutamate in the extracellular space (as detected by microdialysis and microsensors) as well as its origin and function, (Miele et al. 1996;Herrera-Marschitz et al. 1996;Timmerman and Westerink 1997;Jabaudon et al. 1999;Baker et al. 22b;Cavelier and Attwell 25). In the present study potential cellular mechanisms regulating extracellular glutamate were investigated regarding the extent to which each may contribute to glutamate as detected by microdialysis. Given that glutamate is the primary excitatory neurotransmitter and is also involved in various physiological functions such as energy and nitrogen metabolism, makes the physiological interpretation of sampled extracellular levels complex. Glutamate is present in relatively high concentrations in the cytoplasm of all neurons as well as glia cells. Moreover it is released by a number of distinct mechanisms localized in neurons (contributing to synaptic pool) and astrocytes (contributing to extrasynaptic sites). Irrespective of origin, high glutamate concentrations can, however exert stimulation of junctional as well as extrajunctional receptors on both neurons and glial cells influencing glutamate transmission. The existence of the extrasynaptic location of Na + -dependent glutamate transporters, as well as group II and III metabotropic glutamate receptors, which would have functional properties different from those of synaptic receptors in the extracellular space, indicates that extracellular glutamate has ways of autoregulating extracellular glutamate concentrations, which also facilitates different extracellular pools (Baker et al. 22b). The relationship between synaptic and nonsynaptic glutamate to regulate synaptic plasticity has been termed glutamate homeostasis (Kalivas 29). Synaptic or extrasynaptic pools are often described as neuronal, whereas astrocytic pools are considered as metabolic. However this discrimination is certainly too simplistic when astrocytes are able to modify glutamate neurotransmission. It is unknown at present which pool is sampled by the microdialysis probe, but current results which lead us to believe that basal extracellular glutamate levels measured with microdialysis are more related to astrocytic events (>6%), rather than to neuronal impulse flow (Westerink et al. 1988;Moghaddam 1993;Morari et al. 1993;You et al. 1994;Herrera-Marschitz et al. 1996). It has been reported that a major part of extracellular glutamate in dialysis samples is derived from cystine-glutamate exchangers localized on glial cells (Baker et al. 22a). 121

25 Chapter 5 Recent studies have focused on the different mechanisms of glutamate release from glial cells (Jabaudon et al. 1999;Baker et al. 22b;Cavelier and Attwell 25;Le Meur et al. 27). Cavelier and Attwell (25) studied four astrocyte release mechanisms: prostaglandin- and Ca 2+ -dependent release, swelling-activated anion channels, gap junctional hemichannels and P 2 X 7 receptors. However, none of these mechanisms contributed to tonic glutamate. Despite these findings astrocytes seem to regulate tonic levels of extracellular glutamate to some degree. In the present study a dual-probe microdialysis approach in the mesolimbic dopamine pathway was used, in awake freely moving animals, in an attempt to distinguish glutamate s role as a neurotransmitter from its role in non-neuronal events. In this model one probe is inserted into the dopamine cell body region (the VTA) and a second probe is inserted into the axon terminal region of the ipsilateral NAc. Glutamate levels in the VTA are modified by infusing glutamate related compounds in the first probe, whereas their effects on dopamine neurons are recorded by dialysate levels of dopamine from the second probe. By monitoring glutamate in the VTA and dopamine in the ipsilateral NAc, the correlation between extracellular glutamate levels in the VTA (as measured in dialysates) and functional effects exerted on VTA dopamine neurons can be studied. In a control experiment the dopamine neuronal activity within the somatodendritic region of the VTA was inhibited after TTX infusion, which decreased the dopamine output in the terminal region of the NAc to an expected level below 2% of basal dopamine levels, confirming previous studies (Schilström et al. 1998;Mathé et al. 1999;Legault et al. 2). These results showed that the probes were implanted in the desired positions. 4.1 Infusion of glutamate or blocking high affinity glutamate transporters (by infusing TBOA or TBOA combined with glutamate) Even though it is not surprising that glutamate concentrations in the VTA dialysates increased to about 5% of basal glutamate levels after infusion of glutamate in the VTA, in a dose dependent manner, it is interesting that exogenous glutamate ( µm, 1 mm and 1 mm) infused in the VTA did not alter dopamine levels in the ipsilateral NAc. This observation can be explained by rapid uptake of glutamate from the extracellular space by high affinity transporters (Garthwaite et al. 1992;Obrenovitch et al. 1994), which may also explain why glutamate levels do not increase 1 fold with a 1 fold increase in dose. The uptake is apparently so efficient that the glutamate receptors on dopamine neurons are not reached. It can be concluded that concentrations of glutamate injected exogenously cannot be correlated with those of glutamate levels in the synaptic cleft. In 122

26 Are brain extracellular glutamate levels recorded by microdialysis related to synaptic events? line with this observation is the finding that high extracellular concentrations of glutamate, equivalent to those associated with ischemia or traumatic brain injury (TBI), are surprisingly well tolerated in vivo (Obrenovitch et al. 2). Maintenance of the extracellular glutamate concentrations is primarily achieved by Na + - dependent high-affinity glutamate transporters [excitatory amino acid transporters 1 5 (EAAT1 5)]. EAAT1 and EAAT2 have been localized on astrocytes although they have also been observed in neurons, while EAAT3 is located on neurons only (Montiel et al. 25). Therefore, EAAT s are probably responsible not only for the removal of glutamate, termination of glutamate receptor activation, maintenance of low extracellular glutamate concentrations but also for modulation of synaptic transmission (Shimamoto 28). In the next experiment we blocked glutamate transporters by infusing DL-TBOA. DL- TBOA is a non-transportable blocker of Na + -dependent glutamate transporters, that blocks GLAST/EAAT1, GLT-1/EAAT2, EAAC/EAAT3 (Shimamoto et al. 1998;Shimamoto et al. 2). TBOA perfused at increasing concentrations was shown to increase glutamate levels concentration dependently in vivo (Xi et al. 22;Baker et al. 22b;Xi et al. 23;Melendez et al. 25;Day et al. 26), as well as in in vitro experiments (Jabaudon et al. 1999;Angulo et al. 24;Melendez et al. 25;Cavelier and Attwell 25). In the present study TBOA infused in the VTA induced a significant concentrationdependent elevation (to 518% of baseline) in extracellular glutamate in the VTA dialysates. In contrast, only a moderate increase was noticed in extracellular dopamine in the ipsilateral NAc (to 157% of baseline). The dose effect curve shows that the effect was saturated at 5 µm. The TBOA-induced increase in dopamine concentrations in the ipsilateral NAc was accompanied by an increase in stereotypic behaviour; the animals showed intense exploratory activity, rearing, sniffing, licking and ipsiversive turning which are similar to those induced by NMDA infusion into the VTA. The behavioural response and the increase in dopamine release in the NAc demonstrated that glutamate enhanced by TBOA was able to reach its receptors. The dopamine increase in NAc induced by TBOA may be somewhat enhanced by the observed stress response observed (Liaw et al. 25). In order to determine whether infused glutamate can reach its synaptic receptors when the high affinity uptake was blocked, an experiment with increasing concentrations of glutamate in the presence of µm TBOA was carried out. In a previous experiment 123

27 Chapter 5 TBOA ( µm) showed an increase (±268%) in glutamate levels in the VTA but no effect (±15%) in dopamine levels in the NAc. In the combined experiment the increase of glutamate induced by TBOA with glutamate in the VTA was not different from the results obtained with the single glutamate administration. However, the combined treatment of TBOA and glutamate now induced an increase in extracellular dopamine to about % of control in the NAc, indicating that in the presence of TBOA - exogenously administered glutamate could at least partly reach the synaptic glutamate receptors. It is emphasized that other non-blocked uptake mechanisms may still play a role in removing the administered glutamate. 4.2 Modifying astrocytic glutamate release by blocking glutamine synthesis The astrocytic contribution of glutamate to extracellular glutamate levels was evaluated with the dual-probe microdialysis model by inhibiting the astrocyte specific enzyme glutamine synthetase, which converts glutamate into glutamine (Ottersen et al. 1992). This inhibition may lead to decreased glutamine levels and subsequently as glutamine is a precursor for the resynthesis of glutamate to decreased levels of the neurotransmitter. We infused the synthetase inhibitor MSO in the VTA and indeed observed a prounounced and significant decrease (to 42% of controls at the highest infused dose) in extracellular glutamate concentrations in the VTA and a small but not significant decrease of dopamine (to 95% of baseline) in the NAc. The observed decrease in glutamate is in line with a previous study where MSO was infused (Rodríguez Díaz et al. 25). At a high concentration (25 mm) MSO increases dopamine to ±128% of controls (data not shown). The latter observation can be explained by the strong behavioural activity that was induced by the infused drug. MSO is known to potentially cause convulsions in vivo, through a mechanism which is still debated (Cloix and Hevor 1998), however, in this study no convulsions were observed. It is concluded that MSO affected a glutamate pool that is clearly detectable in dialysates. It is likely that this glutamate pool is derived from glial cells but as the dopamine cells were not affected, is apparently not related to synaptic activity. From this observation we may hypothesise that an important part of the glutamate sampled by microdialysis is not directly related to synaptic events. 4.3 Modifying the glutamate-cystine exchanger Glutamate release coupled to the cystine-glutamate exchanger (antiporter) referred to as - system X c has been thoroughly investigated (Baker et al. 22a;Melendez et al. 25;Cavelier and Attwell 25). Cystine-glutamate exchangers are present at a density 124

28 Are brain extracellular glutamate levels recorded by microdialysis related to synaptic events? which is 1-75% of that of Na + -dependent transporters (Anderson et al. 199), suggesting that they have a contributing effect on the extracellular glutamate concentrations. This antiporter is a plasma membrane bound Na + -independent glutamate transporter, which can operate in the reverse mode, i.e. one extracellular cystine molecule exchanged for one intracellular glutamate. The accumulated cystine is reduced to cysteine and in turn converted to glutathione (Warr et al. 1999;Danbolt 21;Baker et al. 22a;Xi et al. 23;Chen and Swanson 23). Thus, rather than eliminating extracellular glutamate, X - c- is a nonvesicular source of extracellular glutamate (Cho and Bannai 199;Patel et al. 24). The antiporter is ubiquitously distributed on cells throughout the body, although in the brain it may be preferentially located on glia cells (Danbolt 21). Baker et al (22) have calculated that (in the NAc) at least 6% of extracellular glutamate in dialysis samples is derived from cystineglutamate exchangers localized on glial cells. In this study the cystine-glutamate exchanger is further investigated with aid of the dualprobe microdialysis approach in the mesolimbic pathway. To determine whether cystine does induce an increase in extracellular glutamate and whether this source of glutamate is able to reach the glutamate receptors on dopamine neurons, cystine was infused at high concentrations in the VTA. At a concentration of µm or 1 mm cystine induced an increase in extracellular glutamate of ±18% and ±196% in the VTA, respectively and an increase in extracellular dopamine of ±126% and ±13% in the NAc, respectively. These results confirm the hypothesis of Baker, indicating that functional glutamate release is possibly coupled to cystine-glutamate antiporter. Next HCA, a non-transportable cystine-glutamate exchange blocker, was infused into the VTA to determine whether the X - C system contributes to extracellular glutamate release. HCA (infused in a concentration of 5 µm) indeed showed a slight but statistically significant decrease in extracellular glutamate levels in the VTA to about 81% of baseline. Dopamine levels in the NAc did not change, however the expected decreases of dopamine might be too low to observed because of the sensitivity of the dual-probe model. Another non-transportable cystine-glutamate exchanger blocker CPG was infused into the VTA (Ye et al. 1999;Patel et al. 24). CPG induced a slight but significant decrease to 85% of baseline in extracellular glutamate levels at the lowest concentration infused (5 µm). However, higher dosages of CPG (5 and 5 µm) induced a (non significant) increase in the extracellular glutamate concentration. CPG did however induce a 125

29 Chapter 5 significant increase (±147% of baseline) in extracellular dopamine levels in the NAc after infusion of 5 µm CPG in the VTA. The observed increases in glutamate and dopamine concentrations may also be explained by non-specific effects of CPG, as the compound has been described as an antagonist for mglur1 and mglur5 and an agonist for mglur2 metabotropic glutamate receptors (Hayashi et al. 1994;Kingston et al. 1995;Patel et al. 24). 4.4 Modifying mglur2/3 receptors by infusing mglur specific compounds Metabotropic glutamate receptors (mglur), unlike receptors for monoamines do not mediate but rather modulate brain excitability via presynaptic, postsynaptic and glia mechanisms (Conn and Pin 1997;Folbergrová et al. 25). mglu2/3 receptors are predominantly localized at the periphery of the presynaptic area, and to a lesser extent postsynaptically (Moldrich et al. 23). Subtype mglur3 is also found on astrocytes (Smolders et al. 24;Rae et al. 25). As they have a high affinity for glutamate, they function to monitor glutamate that has escaped from the synaptic space ( spillover ) and diffuses to perisynaptic sites. Apart from effects on astrocytes, mglur2/3 agonists are believed to reduce glutamatergic transmission by stimulating presynaptic autoreceptors (Moghaddam and Adams 1998;Anwyl 1999;Cartmell and Schoepp 2;Xi et al. 22). mglu2/3 receptors are expressed in the corticolimbic brain, including the VTA and the NAc (Ohishi et al. 1993a;Ohishi et al. 1993b;Tamaru et al. 21;Richards et al. 25). In previous single-probe-microdialysis studies.5-5 µm APDC induced a decrease in glutamate concentrations in both the NAc and PFC (Baker et al. 22b;Melendez et al. 25). In the present study the selective and relative potent group II mglur agonist APDC induced a significant decrease (to ±72% of baseline at the highest dose of 5 mm) in extracellular glutamate levels in the VTA. In line with the expectation, only infusion of this dose of APDC induced a slight but significant decrease of dopamine (to 83% of baseline) in the NAc. Conversely the selective mglur2/3 antagonist APICA caused a significant increase (to ±164% of baseline) of the extracellular glutamate levels in the VTA. Equally, APICA induce a significant increase in extracellular dopamine levels (to ±146% of baseline) in the NAc when 1µM -1mM was infused in the VTA. It is speculated that in these experiments a small but significant pool of glutamate in dialysates was detected that was able to affect the activity of the dopamine neurons. 126